1. Field of the Invention
The present invention relates to a biosensor for quantitating a biochemical substrate (specific compound) contained in a sample liquid such as whole blood, urine, fruit juice and the like, with accuracy, speed and ease, and a method for quantitating a biochemical substrate by the biosensor. More particularly, the invention relates to a biosensor for electrochemically quantitating a concentration of a biochemical substrate in a sample liquid by reacting biochemical substrates such as glucose or cholesterol with an oxidoreductase which can react with specificity to the biochemical substrates, and a method for quantitating a biochemical substrate using thereof.
2. Description of the Related Art
Recently, biosensors have been proposed which can easily quantitate a specific compound (biochemical substrate) in a sample liquid such as a biological sample or a food without diluting or stirring the sample liquid.
For example, Japanese Laid-Open Patent Publication No. 3-202764 discloses a biosensor including an electrode system formed on an insulating substrate by screen printing or the like, a reaction layer formed on the electrode system and a space provided as a sample supply path by using a cover and a spacer. The reaction layer contains a hydrophilic polymer, an oxidoreductase, and an electron acceptor. Such a biosensor can quantitate the concentration of a biochemical substrate in a sample liquid as follows: First, the sample liquid is dropped on the space in the biosensor so as to be supplied to the reaction layer due to capillary phenomenon, thereby dissolving the reaction layer. This causes an enzyme reaction between the biochemical substrate in the sample liquid and the oxidoreductase in the reaction layer, whereby the electron acceptor in the reaction layer is reduced. After the completion of the enzyme reaction, the reduced electron acceptor is electrochemically oxidized, whereby the concentration of the biochemical substrate in the sample liquid is quantitated by an oxidation current value.
In the above-described biosensor, potassium ferricyanide is often used as the electron acceptor. Biosensors using potassium ferricyanide have excellent stability, can be produced at a low cost and thus appropriate in terms of mass production. However, the biosensor using potassium ferricyanide as the electron acceptor is associated with a second-order reaction velocity between the potassium ferricyanide and the oxidoreductase that is slower than that of a biosensor using an electron acceptor such as quinone derivatives or another metallic complex which is unstable or produced at a high cost. Accordingly, in the biosensor using potassium ferricyanide, an enzyme reaction takes substantially long time, thereby causing a problem of not being able to promptly quantitate the concentration of the biochemical substrate.
Additionally, several biosensors are known which employ ferrocene as an electron acceptor which has a faster second-order reaction velocity than those of potassium ferricyanide or derivatives thereof.
Japanese Laid-Open Patent Publication No. 2-240555 discloses a glucose sensor including a working electrode having a photo-curing resin film containing a ferrocene compound and a photo-curing resin film containing glucose oxidase sequentially provided on-the surface of the working electrode. Japanese Laid-Open Patent Publication No. 2-99851 discloses a glucose sensor including a working electrode having a ferrocene compound-containing layer and a glucose oxidase-immobilized layer on the ferrocene compound-containing layer on the surface of the working electrode. In the above-mentioned biosensors, 1,1xe2x80x2-dimethyl ferrocene, ferrocene, i.e., bis(cyclopentadienyl)iron(II), vinyl ferrocene or the like is used as the ferrocene compound.
Japanese Laid-Open Patent Publication No. 5-256812 discloses a glucose sensor including a layer carrying glucose oxidase and a ferrocene compound on a working electrode and a means for maintaining a predetermined temperature in the vicinity of the working electrode. In this biosensor, ferrocene and/or derivatives thereof is used as the ferrocene compound.
However, any ferrocene compound used in the above-described glucose sensors normally exists as a reduced form. Therefore, in order to achieve electron transfer from the biochemical substrate to the working electrode by an enzyme reaction, the ferrocene compound needs to be converted into oxidized form on the electrode.
Japanese Laid-Open Patent Publication No. 6-3316 discloses a glucose sensor (a modified electrode) modified by a hydrophobic redox substance (e.g., a ferrocene) which has been ionized in advance in an aqueous solution, and a hydrophilic enzyme (e.g., glucose oxidase) on a surface of a conductive electrode. The glucose sensor is produced as follows: Ferrocene is electrolyzed in a phosphate buffer to form a solution containing ferricinium ions. Then, a glucose oxidase is added to the solution. The resultant mixed solution is applied to or electrodeposited on the surface of the conductive electrode.
However, the above-described modified electrode has a problem in that the phosphate ion and the ferricinium ion form an ion-like complex which renders the surface of the electrode inactive. Moreover, a step of electrolyzing ferrocene is required in order to produce the modified electrode. As a result, increased production time and increased cost are required.
According to one aspect of the present invention, a biosensor includes: an insulating substrate; an electrode system formed on the insulating substrate which has a working electrode and a counter electrode; and a reaction layer formed on the insulating substrate which contains an oxidoreductase and an electron acceptor. The electron acceptor is ferricinium ion derived from ferrocene electrolyte.
In one embodiment of the present invention, the ferrocene electrolyte is selected from the group consisting of ferrocenium hexafluorophosphate and ferrocenium tetrafluoroborate.
In one embodiment of the present invention, the reaction layer further comprises at least one surfactant.
In one embodiment of the present invention, the reaction layer further comprises at least one hydrophilic polymer.
In one embodiment of the present invention, the oxidoreductase is selected from the group consisting of glucose oxidase; glucose dehydrogenase; lactate oxidase; lactate dehydrogenase; uricase; cholesterol oxidase; a combination of cholesterol oxidase and cholesterol esterase; a combination of glucose oxidase and invertase; a combination of glucose oxidase, invertase and mutarotase; and a combination of fructose dehydrogenase and invertase.
According to another aspect of the present invention, a method is disclosed for quantitating the concentration of a biochemical substrate in a sample liquid by using a biosensor including an insulating substrate, an electrode system formed on the insulating substrate which has a working electrode and a counter electrode and a reaction layer provided on the insulating substrate which contains an oxidoreductase and an electron acceptor. The method includes the steps of: adding the sample liquid to the reaction layer; and detecting a response current value by applying a voltage between the working electrode and the counter electrode. The electron acceptor is ferricinium ion derived from ferrocene electrolyte.
Thus, the invention described herein makes possible the advantages of (1) providing a biosensor for promptly quantitating a concentration of a biochemical substrate with sufficiently short enzyme reaction time; (2) providing a biosensor for quantitating a concentration of a biochemical substrate with accuracy without deteriorating a detecting sensitivity; (3) providing a biosensor produced at sufficiently low cost; and (4) providing a method for quantitating a concentration of a biochemical substrate using the above-mentioned biosensor with accuracy and speed.
These and other advantages of the present invention will become apparent to those skilled in the art upon reading and understanding the following detailed description with reference to the accompanying figures.